1. Technical Field
The present invention is directed to an improved method of making starting materials involved in the preparation of an assay for determining the presence and/or amount of the immunosuppressant drug, FK506, in human blood. More specifically the present invention is directed to the preparation of reaction products of FK506 with dicarboxylic acids or anhydrides thereof.
2. Background
FK506 is an immunosuppressant useful for the treatment of rejection following transplant surgery, graft versus host disease and autoimmune diseases in humans. FK506 is a macrolide antibiotic isolated from the fungus Streptomyces tsukubaensis by the Fujisawa Pharmaceutical Company of Japan. Cyclosporine, another immunosuppressant (but having a totally different structure from FK506), has also been used to control rejection. During cyclosporine therapy, monitoring the blood concentration of cyclosporine is an important aspect of clinical care. Accordingly, it is expected that monitoring blood concentrations of FK506 will be important for patients receiving this drug.
To accurately and precisely measure blood concentrations of FK506, an appropriate analytical method must be available. High performance liquid chromatography (HPLC) is one non-immunological method that could be utilized. A receptor based assay could be configured using FK-binding protein and any number of reagents to generate a signal. Numerous immunological configurations can also be envisioned which could be successfully applied to the measurement of FK506.
EP 0 293 892 A2 describes an ELISA methodology to measure FK506 comprised of 1) an ELISA plate coated with anti-FK506 antibodies, 2) an FK506-horseradish peroxidase conjugate which competes with free FK506 and acts as a signal generating reagent and 3) an appropriate substrate for the peroxidase. EP 0 293 892 A2 teaches that an immunogen generally is utilized in the form of a conjugate of FK506 with a carrier such as bovine serum albumin (BSA) by converting the FK506 to a half ester of a dicarboxylic acid such as succinic acid, then reacting the half ester with N-hydroxysuccinimide or the like in the presence of a condensing agent such as dicyclohexylcarbodiimide and further reacting the resulting activated ester with BSA. Pages 6 and 7 of the publication disclose the preparation of the FK506 hemisuccinate utilizing pyridine. However, it has been found that utilization of pyridine tends to result in the formation of disadvantageously low amounts of the product, FK506 half-ester. This tendency has been found, for example, when succinic anhydride has been utilized as the dicarboxylic acid. The achievement of consistently reliable and repeatable yields of reasonable amounts of FK506 half-ester product clearly is desirable. Moreover, the method disclosed in EP 0 293 892 results in undesirable amounts of FK506 being converted into side products thereby reducing the amount of unreacted FK506 that can be recovered following reaction.
The production of low yields of FK506 hemisuccinate coupled with the undesirable amount of side reaction products of FK506 is economically disadvantageous when amounts of FK506 hemisuccinate necessary for commercial scale production are contemplated. Accordingly, an object of the present invention is to provide an improved method for producing esters of FK506 and dicarboxylic acids which reliably will yield improved amounts of the product half-esters of FK506 and, at the same time, reduce the amount of FK506 converted into undesirable side products.